This invention relates generally to analysis of cellular metabolites that contain, or can be induced to contain one or more charges. More particularly, this invention relates to a shotgun metabolomics approach that exploits multiple combinatorial sets of engineered stereoelectronic interactions between matrix and analyte to enable high throughput metabolomics directly from extracts of biologic material without the need for prior chromatography. Through sequential matrix assisted laser desorption/ionization (MALDI) sets of chemically related metabolites can be selectively ionized, identified by tandem mass spectrometry and quantified. The novel properties of engineered analyte/matrix interactions containing resonance-stabilized delocalized charge allow the high throughput identification and quantitation of many hundreds of metabolites directly from extracts of biologic materials without prior chromatography. This approach has general utility for the determination of the chemical composition of complex mixtures of metabolites from biologic tissues, biologic fluids (e.g., blood, urine, cerebrospinal fluid) as well as metabolic flux measurements and pharmacokinetics through ratiometric comparisons with stable isotope standards.
Metabolomics is an emerging field that provides critical insight into the physiologic status of cells by identifying and quantifying multiple cellular metabolites. As a complement to genomics, proteomics, and transcriptomics, metabolomics has been successful in discriminating a wide variety of different metabolic phenotypes where more conventional assessments have failed. Through assessment of alterations in the profiles of metabolites, new insights into disease processes have already been made.
Among the various technologies that have traditionally been employed to identify and quantify cellular metabolites, mass spectrometry (MS) has evolved to be a very powerful tool for metabolite analysis. The high sensitivity and resolution of MS, in conjunction with its ability to elucidate the structure of unknown compounds present in complex biological samples, have provided a strong impetus to use MS in the analysis of metabolites present in low abundance (i.e., approximately less than about 0.1% of total content) and extremely low abundance (i.e. approximately less than about 0.01% of total content). However, the use of mass spectrometry in metabolomics has been severely limited by the difficulty in developing conditions for the ionization of large numbers of metabolites containing greatly differing chemical functionalities. In part, this previously intractable problem was due to the absence of an approach which provided rapid access to suitable combinations of ionization conditions that could be effectively multiplexed into a set of combinatorial conditions that facilitate selective ionization of the diverse sets of chemical functionalities present in metabolomes of biologic materials (e.g., tissues and fluids). Moreover, identification of mass alone does not establish the chemical structure of a metabolite due to the large number of isomeric metabolites present in nature. The use of tandem mass spectrometry, or other approaches, is necessary to identify the structure components and the isomeric composition of the analytes of interest.
Gas chromatograph mass spectrometry (GC-MS) has been widely used in the analyses of volatile metabolites or metabolites that can be volatized after chemical derivatization. Electrospray ionization mass spectrometry (ESI-MS) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) can be coupled to liquid chromatography (LC) or capillary electrophoresis (CE), allowing high-throughput analyses of nonvolatile metabolites from biological materials. These techniques have been recently reviewed and represent valuable tools for metabolomics research. The use of chromatography or electrophoresis, however, extends analysis time and introduces additional procedural complexity. Quantitative analysis of the chemically diverse molecular species present in the metabolome often requires multiple different chromatographic approaches to resolve or enrich salient metabolites. Current efforts in metabolomics are thus aimed at maximizing the amount of information obtained, while minimizing time and methodological difficulty necessary for sample analysis.
Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), and in addition, MALDI-tandem mass spectrometry, has now matured and are widely used in proteomics analysis, nucleotide sequencing, and polymer analysis, has the potential to contribute significantly to metabolomics. MALDI-MS permits the discrimination of isomeric molecular species that would not be possible using spectra of molecular ions alone. MALDI-MS has a higher tolerance to salts than ESI-MS and APCI-MS and has the unique ability to generate singly charged ions of less than about 1000 dalton (Da) which can avoid the overlapping of ion peaks produced by multiply charged ions routinely occurring at low m/z values in ESI-MS and APCI-MS.
However, due to the diverse array of functionalities in cellular metabolomes, progress in identifying matrices that provide broad coverage has been rate limiting. In particular, traditionally the use of MALDI-MS has been restricted to the analysis of high molecular weight metabolites because conventional matrix clusters (e.g., cyanohydroxycinnamic acid) create excessive noise in the low-mass range of the spectrum and interfere with the detection of low molecular weight cellular metabolites. Until recently, this has precluded the routine use of MALDI-MS in metabolomics. Accordingly, although, MALDI-MS offers the potential advantage of rapid throughput which, when combined with stable isotope standards for metabolites of interest that can theoretically generate large amounts of quantitative information with unprecedented speed and accuracy, previous work has failed to obtain suitable methods for the effective ionization/desorption of the multiplicity of chemical entities in biologic samples using MALDI ionization/desorption methods. Moreover, in many cases, the use of mass determination alone does not allow the identification of the relative contributions of the chemical structures that are present in an ion peak due to the large number of isomeric metabolites in biologic samples.
Matrices have now been identified, however, that produce minimal spectral noise in the low molecular weight region of interest. Examples include the silicon nanoparticle, mesotetrakis (pentafluorophenyl)porphyrin, 9-aminoacridine, porous silicon, cyanohydroxycinnamic acid, dihyroxybenzoic acid, trihydroxyacetophenone and ionic liquid matrices, all of which have been examined and their utility in specific applications confirmed. Recently, MALDI-MS, employing 9-aminoacridine (9-AA) matrix, was used to “identify” 29 metabolites by mass alone from extracts of the islets of Langerhans. However, this low number of metabolites does not represent sufficient coverage of the cellular metabolome and even the “identified” metabolites contain unknown contributions from different isomeric species. Previously, it was recognized that 9-aminoacridine may be used as a matrix since it produced only small amounts of ion clusters after laser excitation. However, this diminutive coverage, lack of reproducibility between preparations, and the errors made by not knowing isomeric content precluded its effective use in metabolomics investigations. Enabling advances that greatly expand coverage of the metabolome and provide definitive identification of isomeric molecular species through tandem mass spectrometric approaches were necessary for further progress. Thus, prior work neither recognized the vastly expanded information content that could be generated by multiplexing engineered analyte-matrix pairs nor the necessity of tandem mass spectrometry for identification of the structural assignments of isobaric peaks (i.e., peaks having identical nominal masses but not necessarily structural isomers) as well as peaks resulting from chemical isomers (i.e., isomers with exactly the same elemental composition and mass).
We theorized that the facile ability of 9-AA to undergo combinatorial changes in its stereoelectronic states, intermolecular interactions and π stacking will promote a rich repertoire of analyte-matrix interactions to allow extended coverage into the metabolome. Without being bound by theory, we envisaged that combinatorial multiplexing of stereoelectronic interactions may be induced by alteration in charge state modulating the HOMO-LUMO (highest occupied molecular orbital-lowest unoccupied molecular orbital) interactions in the well-suited aromatic matrix 9-AA which contains a primary amine that undergoes pH-induced changes in charge state. The delocalization of electron density and charge in the 9-AA matrix facilitates the development of novel analyte/matrix interactions that can be exploited for ionization and desorption of analytes. In one aspect, these novel interactions can be envisaged to result from analyte interactions with the formal structures of three discretely charged 9-AA matrices containing delocalized electrons as shown schematically in FIG. 1.